Multifunctional tissues must simultaneously execute diverse physiological processes, balancing those processes across the finite number of cells within the tissue and limited capacity for transcription, translation and biochemical reactions within each cell. The constraints imposed by using the same tissue for multiple purposes can result in sub-optimal performance of each process, and it is largely unknown how tissues balance their roles. In this project, we will test how a multifunctional, immunologically active tissue can achieve a robust innate immune response while simultaneously performing other crucial organismal functions. The insect fat body is a multifunctional tissue that can serve as a generic model for how polyfunctional organs achieve diversified tasks, including management of immune response to infection. Fat body functions span those of at least three vertebrate organs: immune system, adipose tissue, and liver. The fat body is the primary systemic immune organ in insects, but also serves as the metabolic control organ responsible for storage and release of lipids and other nutrients and produces most of the yolk used to provision developing eggs. This is analogous to vertebrate adipose tissue, which is a cellularly heterogeneous tissue that stores lipids and also produces cytokines and inflammatory reactions in response to infection. We hypothesize that the multiple functions of the fat body rely on dedicated and differentiated subpopulations of cells within the fat body tissue. We will use single-cell RNA sequencing (scRNAseq) to test the hypothesis of cellular heterogeneity in Drosophila melanogaster (fruit fly) that are either actively mounting an immune response or that remain unstimulated, both while actively engaged in egg development and in the absence of reproductive investment. We will identify diagnostic mRNA transcripts that define cellular subpopulations or transcriptional states and will design fluorescent probes to these in order to facilitate future inexpensive and high-throughput experimentation. Protocols for synthesis and use of these probes will be publicly distributed on the internet in order to enable subsequent study by other research teams. We anticipate that this project will serve as a model for determining how polyfunctional tissues balance competing physiological functions, providing mechanistic understanding for the classical life history tradeoff between immunity and reproduction and generating a community resource that will immediately enable sophisticated study of the D. melanogaster fat body by our group and others. Our findings will apply generally to organisms with multifunctional tissues and specifically to insects of agricultural or public health importance, stimulating analogous research in other insect and animal systems.